Deep ocean water alters the cholesterol and mineral metabolism of squid Todarodes pacificus and suppresses its weight loss

This study is the first to demonstrate that deep ocean water (DOW) has physiological significant effects on squid. After 36 h of rearing squids, those reared with DOW had significantly higher total and free cholesterol levels and lower alanine transaminase activity in hemolymph as compared with those reared with surface sea water (SSW). SSW rearing also resulted in 6.95% weight loss, while DOW rearing caused only 2.5% weight loss, which might be due to liver metabolism suppression. Furthermore, both monovalent (sodium, chloride, and potassium ions) and divalent (calcium, inorganic phosphorus, and magnesium ions) ions in hemolymph were elevated when reared with DOW compared to those when reared with SSW. A study of genes expressed in the brain revealed that five genes were specifically remarked in DOW rearing. Most altered genes were neuropeptides, including those from vasopressin superfamily. These neuropeptides are involved in cholesterol and/or mineral metabolisms and physiological significant effects on squid. This study is the first report the effects of DOW on cholesterol and mineral metabolism of squid and will contribute to squid aquaculture using DOW.

www.nature.com/scientificreports/ levels in nonalcoholic fatty liver disease mice fed with high-fat diet 5 . Based on a mammalian investigation of DOW, DOW influences lipid metabolism and possesses healthy effects. For aquaculture, the growth of seaweeds 6,7 and shrimp 8 was promoted by breeding in DOW as compared with those bred in surface sea water (SSW). The germiling growth rate of brown alga, Sargassum fusiforme, kept with DOW was 2.7 times higher than those kept with SSW 7 . The growth of juvenile sporophytes of Eisenia arborea and Eisenia cava reared with DOW was also faster 6 . Pelagic shrimp Sergia lucens that lives in deep sea can be kept for a long time when reared with DOW 8 ; it could be kept for an average of only 13 days with SSW and 58.8 days with DOW. The shrimp could be kept for up to 185 days with DOW 8 .
Todarodes pacificus (Fig. S1), the Japanese common squid, is distributed in the surface and middle layers of nearshore waters from the Sea of Okhotsk north to the Sea of Japan and East China Sea. This squid is in the highest demand in Japan and Asian region; it is used not only fresh, but also in various processed foods, such as surume (dried squid) and shiokara (salted squid). However, the technology for rearing this squid has not been developed.
Our recent study found that DOW reduced the stress of marine teleost (Japanese flounder Paralichthys olivaceus) which was grown under high density condition 9 . In the study, kynurenine, a component existing in DOW, was identified as the responsible factor for the stress-reducing effect of DOW 9 . These findings suggest the positive effect of DOW on the physiological traits of the squid. To test this possibility, the current study compared changes in hemolymph composition and mRNA expression in the brain, as well as those in the body weight between DOW-and SSW-reared squids in the condition of identical water temperature.

Results
Changes in hemolymph components after rearing squids in SSW or DOW. The total protein (TP), albumin (ALB), and glucose (GLU) levels in squid hemolymph did not change between DOW and SSW rearing ( Fig. 1), while cholesterol metabolism was significantly changed. The total cholesterol (T-CHO) and free cholesterol (F-CHO) levels in the hemolymph of squids reared with SSW were significantly lower than in those reared with DOW, although ester-type cholesterol (E-CHO) did not significantly change (Fig. 1). Triglycerides were not detected in the squid hemolymph at least present conditions. Those reared with DOW had significantly lower hemolymph alanine transaminase activity (ALT) as compared with those reared with SSW ( Fig. 2). No significant difference was found in the hemolymph aspartate transferase (AST) and creatine kinase (CK) activities of squids kept with DOW and SSW (Fig. 2). In addition, changes in body weight before and after rearing in SSW or DOW are shown in Table S1. Interestingly, DOW rearing only caused 2.5% weight loss, while SSW rearing resulted in 6.95% weight loss.
Changes in hemolymph mineral concentrations after rearing squids in SSW or DOW. Monovalent ions (Na + , Cl − , and K + ) and divalent ions (Mg 2+ and Ca 2+ ) in SSW and DOW showed almost the same values (Table S2). However, Na + , Cl − , and K + levels in the hemolymph of squids reared with DOW were significantly higher than in those reared with SSW ( Fig. 3A-C). The concentration of hemolymph Mg 2+ in squid reared with DOW was significantly higher than in those reared with SSW (Fig. 3D). In the case of Ca 2+ , the hemolymph Ca 2+ www.nature.com/scientificreports/ level of squids reared in DOW tended to be higher than those of squids reared in SSW (Fig. 3E). The hemolymph inorganic phosphorus ion (iP) level of squid kept with DOW was significantly higher than in those reared with SSW, as Mg 2+ did (Fig. 3F).
Changes in mRNA expression in the brain after rearing squids in SSW or DOW. Variations in expression (volcano plot) in the brains of squid reared with SSW and DOW are shown in Fig. 4A. Transcript IDs with significant changes between SSW and DDW (LogFC > 5.0 and false discovery rate [FDR] > 10 −6 ) are shown in Fig. 4A. Squid reared in DOW had changes in genes expressed in the brain. Figure 4B shows a heat map with hierarchical clustering obtained by Trinity utility. Based on hierarchical clustering analysis, we found 50 genes whose expression varied significantly by DOW and SSW rearing (Table S3). Among these genes, there were five protein-coding genes whose amino acid-coding regions could be inferred.

Discussion
This study is the first to demonstrate that DOW has physiological significant effects on Japanese common squid T. pacificus. After 36 h of rearing squids, those reared with DOW had significantly higher T-CHO and F-CHO levels and lower ALT activity in hemolymph as compared with those reared with SSW (Fig. 1). The ALT activity, a liver marker [10][11][12] , also decreased in DOW rearing (Fig. 2), suggesting that liver metabolism was reduced and hemolymph cholesterol levels remained high. Additionally, their pre-and post-experimental weights were measured (Table S1). The average weight of nine squids reared with SSW decreased from 148.2 to 137.9 g, while the average weight of those reared with DOW changed from 148 to 144.3 g, indicating a small percentage reduction (− 2.5%) in weight. Those reared with DOW had a reduced weight loss by suppressing liver metabolism. On the other hand, their hemolymph AST and CK levels, which are markers of cardiac and skeletal muscle 11,13-15 , did not significantly decrease, possibly because they were constantly moving their muscles to swim. In this study, DOW rearing affected mineral metabolism in squid. Both monovalent (Na + , Cl − , and K + ) and divalent ions (Mg 2+ and Ca 2+ ) in hemolymph were elevated when reared with DOW compared to those reared with SSW (Fig. 3). Mineral ions other than Ca 2+ were significantly elevated after DOW rearing (Fig. 3). Since Ca 2+ plays an important role in squid neural activity 16,17 , this ion may be regulated by a different mechanism.
A study of genes expressed in the brain revealed that five genes were specifically remarked in DOW rearing (Figs. 4 and 5). Most altered genes were neuropeptides, including oegopressins superfamily, achatin-related peptide, and elevenin-like peptide, implying that they significant physiological effects on squid.
In the Octopus species, two peptides of Octopressin and Cephalotocin including Vasopressin/Oxytocin superfamily have been isolated and identified from the rectum and nervus tissues in Octopus vulgaris, respectively 18,19 . Our determined two peptides belonged to Vasopressin/Oxytocin superfamily (Fig. S2 and Table S4). Sequence alignment by MAFFT showed that our determined peptides were composed of nine amino acid residues containing consensus cysteine residues as well as other bilaterian Vasopressin/Oxytocin peptides (Fig. 6A). Since these types of peptides are the first to be discovered in open-eyed squids (Oegopsids), we name it Oegopressin. The present study is the first report showing the expression of oegopressins in squid. The octopressin and cephalotocin genes, like the Vasopressin/Oxytocin family, were known to have evolved through duplication 20 . Both peptides in this study showed a similar degree of homology compared to the previously known Conopressin (Lymnaea stagnali: No. 1, Fig. 6B). The three previously known Cephalotocins (Nos. 11, 12, and 13, Fig. 6B) have a second phenylalanine and a third tryptophan, but none of the peptides found in this study are identical to these. Therefore, we concluded both novel Todarodes peptides are Octopressin homologs and determined Oegopressin 1: CFFRNCPPG (No. 6, Fig. 6B) and Oegopressin 2: CYFRNCPAG (No. 10, Fig. 6B) in squid. Whether other squid species besides the common squid have a separate Cephalotocin homolog will require further investigation of the genome sequence in more species in the future.
Both coding sequences are characterized by the presence of an additional functional Neurophysin sequence behind the mature peptide (each underline in Fig. 5A). In octopus, Octopus vulgaris, both octopressin and cephalotocin mRNA were expressed in the esophageal brain 19 . This fact is agreement with our RNA-sequencing results. After 1 day administration Octopression into octopus, the hemolymph osmolality and Ca 2+ concentrations decreased 21 . As described above, the fact that only the Ca 2+ in hemolymph, unlike the other ions, was not significantly elevated when reared with DOW may have something to do with the action of Octopression.
Achatin-I, a tetrapeptide (Gly-d-Phe-Ala-Asp), was purified and determined from the suboesophageal and cerebral ganglia of the African giant snail, Achatina fulica Férussac 22 . This peptide had a bioactivity and evoked a potent neuroexcitatory effect, although Gly-l-Phe-l-Ala-l-Asp, termed Achatin-II, was ineffective on the neurons of African giant snail 22,23 . The mRNA expression of this peptide increased in the squid brain when reared with DOW. This is the first report of this peptide in a cephalopod. According to a BLAST search, only eight sequences were deposited; all had amino acid sequences encoding multiple peptides, and the sequences of mature peptides were polymorphic with GSWN or GSWD, which is also the case for squid (Figs. 5B and 7). The one coding sequence encoded six mature peptides, whereas the others encoded four to five peptides, and the peptidase excision sites were also conserved (Fig. 7). We intend to investigate the presence of D-type amino acid residues in this peptide and its bioactivity in detail.
Elevenin was identified as a cDNA sequence encoding a neuropeptide precursor from the L11 neuron in the abdominal ganglia of California sea hare Aplysia californica 24 . Thereafter, the knockdown of Elevenin by RNA interference caused severe cuticle melanization in the brown planthopper Nilaparvata lugens 25 . Furthermore, the administration of synthetic Elevenin peptide rescued the body color phenotype in Elevenin-dsRNAi-treated individuals and suppressed the melanization of black insects grown under natural conditions 25 . An Elevenin-like peptide (CKVFIFHPKCRGVAA) found in the squid brain may be involved in melanin metabolism in squid. This peptide codes a single mature peptide like Oegopressin 1 and 2 (Fig. 5). According to a BLAST search, 12 sequences were deposited. There was a variation in the sequence length of the mature peptide, but the consensus cysteine residues were well-conserved (Fig. 8A,B).
It is known that the Vasopressin/Oxytocin superfamily regulates mineral metabolism [26][27][28] . Several peptides in invertebrates are also involved in the regulation of lipid metabolism 29,30 . Thus, these peptides upregulated in the squid brain after rearing with DOW are likely to have a physiological activity in squid and regulate both mineral and lipid metabolism. In mammals, Mg 2+ in DOW has an important role in lipid metabolism 1,5 . In mammals, brain neuropeptides may also be involved in lipid metabolism regulation by DOW. The analysis of the actions of these peptides in squid may also contribute to the effects of DOW on lipid metabolism in mammals. Thus, we would like to investigate the effects of these peptides on squid to determine their physiological effects in squid and contribute to squid aquaculture.
One important issue we raised was the mechanism underlying DOW influences the physiological traits of the squid. We have found that DOW reduced stress in marine teleosts which were grown under high density

Conclusion
DOW has significant physiological effects on T. pacificus. Those reared with DOW had a reduced weight loss as compared with those reared with SSW. Thus, the achievement of our research using DOW could be applied to squid rearing techniques.

Materials and methods
Statement on the ethical treatment of animals. This study has been conducted in compliance with recommendations of the ARRIVE Guideline 31 for reporting in vivo experiments with research animals. All experimental protocols in this study were approved by the Animal Welfare Committee of Kanazawa University. All experiments were performed in a manner that minimized pain and discomfort.
Animals. Japanese common squid T. pacificus (n = 19, 148.1 ± 5.4 g) were collected in Toyama Bay by a fisherman. To confirm the squid species, the COI gene (TRINITY_DN15407_c0_g2_i1) was cloned from the collected squid. Sequence of the cloned gene was then determined to conduct a BLAST search. As the result, the deter-  Rearing squid with DOW or SSW. The squids were divided into two groups (SSW: n = 9; DOW: n = 10) and kept with SSW or DOW for 36 h at 15-16 °C. These squids were not fed bait. After rearing with SSW or DOW for 36 h, these were anesthetized with cold seawater, and hemolymph was taken from their branchial heart using a syringe. The collected hemolymph was put into a 1.5-mL tube. Then, the tube was centrifuged at 5200×g for 5 min. The separated hemolymph was immediately frozen and kept at − 80 °C until further use. After hemolymph sampling, each squid was dissected. The brain above the esophagus was extracted, placed in RNAlater (Sigma-Aldrich, St. Louis, MO, USA), and stored at − 80 °C. Additionally, changes in body weight before and after rearing were examined. Since this species cannot be reared individually, changes in the average body weight of the SSW and DOW groups were calculated instead using their individual body weight.

Measurement of mineral concentration and hemolymph components. Hemolymph samples
were sent to a commercial vendor (Oriental Yeast Co., Ltd., Tokyo, Japan), and Na + , Cl − , and K + were measured through an ion electrode method using a Hitachi 7180 automatic analyzer (Hitachi High Technologies Corporation, Tokyo, Japan). Hemolymph Mg 2+ , Ca 2+ , and iP levels (mg/dL) were determined using assay kits (Mg 2+ : Mg·N, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan; Ca 2+ : Ca II, Shino-Test Corporation, Tokyo, Japan; iP: IP-II, Kyowa Medex Co., Ltd., Tokyo, Japan). TP, ALB, GLU, T-CHO, F-CHO, E-CHO, triglyceride, AST activity, ALT activity, and CK in hemolymph were measured using several kits (FUJIFILM Wako Pure Chemical Corporation). www.nature.com/scientificreports/ Analysis of mRNA expression in the brain after rearing squids with SSW or DOW. Total RNAs were isolated using a kit (RNeasy Plant Mini Kit, Qiagen GmbH, Hidden, Germany). Genomic DNA was removed using an RNase-Free DNase Set (Qiagen). A complementary DNA library was constructed and sequenced with a 150 bp paired-end module using Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA). Raw sequence reads were deposited at the DNA Data Bank of Japan (DDBJ) under the DDBJ Sequence Read Archive (DRA) accession no. DRA015361. Adaptors and low-quality reads were removed using fastp v0.23.2 (default setting 32 ). Subsequently, unigenes were obtained using Trinity assembly program v2.8.5 33 . Only contigs with transcript per million greater than 1.0 were filtered with Trinity utility v2.14.0 and used for subsequent analysis. Kallisto was used for mapping analysis 34 . Statistical analysis for differentially expressed genes was performed with edgeR in the Trinity utilities. Transdecoder v5.5.0 was used to estimate gene-coding regions (https:// github. com/ Trans Decod er/ Trans Decod er), and eggNOGmapper v2.1.9 was used for the functional annotation of amino acid sequence data 35,36 . Homologous sequences of neuropeptide sequences (oegopressin 1 and oegopressin 2, achatin-related peptide, and elevenin-like peptide) were estimated by NCBI webBLAST (blastp) (as of November 27. 2022). Alignments of amino acid sequences were estimated with MAFFT on EMBL-EBI 37 . Mview on EMBL-EBI was used to reformat the results of MAFFT alignment. Sequence logos were generated using Weblogo3 (https:// weblo go. three pluso ne. com/) to show the sequence conservation at each sequence position 38,39 . Statistical analysis. All results are expressed as means ± standard error. The statistical significance between the control and experimental groups was assessed using an independent sample t test. The selected significance level was p < 0.05.